Structural Studies of Alix and ESCRT Complexes in HIV-1 Budding HIV-1 particle assembly and release depend on a protein network that includes Alix and Vps4A/B, and four multiprotein complexes: Hrs/STAM and ESCRT-I, II, and III. These proteins and complexes are conserved from yeast to human, and their normal function is to sort monoubiquitinated receptors, enzymes, and other cargo to the lysosome or vacuole. Inactivation of any one of several proteins tested in this network blocks HIV release and infectivity. The objectives of this projects are to 1) map the molecular interactions between HIV and ESCRT components at the level of individual protein domains;2) characterize the binding affinities and relevant structures of the components involved in these interactions;and 3) in collaboration with Eric Freed, NCI, to design means for inhibiting HIV-1 budding from cells. Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. In this FY we reported the 1.4 to 1.6 structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main-chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions. We went on to determine the structure of modified peptidomimetics and used the structures to explain the gain in affinity from additional designed-in interactions. The normal function of the ESCRT-0 and ESCRT-I complexes is to coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The structures of the human TSG101 UEV domain bound to the peptides described above led to a conundrum about the evolution of the UEV domain. The 1.85 crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centered on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is nonessential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.